The publications and other materials used herein to illuminate the background of the invention and, in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Lanthanides and their chelates have become an important group of labels in various assays, such as immunoassays, hybridization assays, receptor-ligand assays and others [reviews: Hemmilä, Application of Fluorescence in Immunoassays, Wiley, 1991; Hemmilä, St{dot over (a)}hlberg and Mottram (eds.), Bioanalytical Applications of Labeling Technologies, Wallac, 1995; Hemmilä and Mukkala, Crit. Rev. Clin. Lab. Sci., 38(6): 441–519 (2001)]. The long excited state lifetimes of lanthanides makes it possible to exploit very efficiently and simply time-resolution in order to get rid of background interferences and to obtain ultimate sensitivities of fluorometry. Other advantages of lanthanide labels relate to their exceptional spectral properties such as long Stokes' shifts (over 250 nm) and narrow banded ion-characteristic emission lines. The spectral properties allow lanthanides to be used in real multi-label assays where the detection can take advantage of both spectral and temporal resolutions.
There are numerous technologies using lanthanides as labels. The first and the original technology, i.e. the DELFIA® technology, uses two chelate systems, one optimized for labeling and the second, which is created after the actual assay is accomplished, to enable fluorescence enhancement and detection (U.S. Pat. No. 4,565,790, EP 0 064 484). This technology is still the most sensitive and widely used. It has many applications in diagnostics, screening, drug discovery and other research areas. Regardless of an extensive search and numerous patents, development of a single lanthanide chelate structure with optimized properties allowing similar assay performances without enhancement has remained a challenge, due to e.g. energy transfer, intensity, protection, biocompatibility and coupling problems.
A major problem with the original DELFIA® technology relates to the change in ligand of the enhancement process. Using existing enhancement composed of trifluoro derivatives of β-diketones, most commonly naphthoyltrifluoroacetone (β-NTA, i.e. 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione), the lowest pH one can use is about 3.0. A special protein labeling chelate for that purpose has therefore been developed based on diethylenetriamine-N,N′,N″,N″-tetraacetate group (DTTA) (U.S. Pat. No. 4,808,541, EP 0 139 675), which at the pH applied in the enhancement solution rapidly releases europium ions and creates new, highly fluorescent chelates with naphthoyltrifluoroacetone present in excess in the enhancement solution.
When a bioassay requires a labeling chelate of higher thermodynamic or kinetic stability, the original enhancement system requires considerably longer time for enhancement, which is not convenient or even acceptable when rapid universal systems are to be developed. For example, the use of DNA probes quite often require a more stable labeling chelate reagent and for DNA based applications lanthanide chelate of 2,2′,2″,2′″-[[4-[2-(4-isothiocyanatophenyl)ethyl]-pyridine-2,6-diyl ]bis(methylenenitrilo)]tetrakis(acetic acid) (EP 0 298 939, U.S. Pat. No. 6,127,529) is found optimal. The chelate used, however, requires longer fluorescence development times and routinely 20–30 minutes are required to stabilize the fluorescence in the enhancement solution.
An application where the present DELFIA® technology has been found unsuitable is rapid random access analysis of samples performed with all-in-one dry reagent system [Pettersson et al., Luminescence, 15:399–407 (2000)]. In this system the drying procedure used to prepare dry-reagent assay-specific all-in-one wells requires a strong chelating reagent due to risk of ion dissociation during the drying process. Use of DATA chelates optimized for DELFIA® have not been found very suitable for this approach. On the other hand, when more strongly chelating labeling reagents are used, the time required for enhancement becomes too long for the whole process.
Another case where original DELFIA® technology is not suitable is the analysis of plasma samples that may contain high concentration of citrate or EDTA. In one-step assays of analytes (required e.g. in competitive analysis of haptenic antigens) the DELFIA-optimized DTTA chelate can not be used due to the competing chelation processes.
Yet another assay-type requiring improved enhancement/labeling system relates to applications where either free or complexed lanthanides are used as labels. Example of these assays is e.g. cytotoxicity assays, where europium chelate of DTPA is used as intracellular label. Other resembling assays can use lanthanide as labels/tracers for a wide variety of processes (environmental samples, metabolic routes etc.).
A further assay-type requiring label chelates with higher stability than that of DTTA and improved enhancement relates to applications where the reaction mixture contains high concentrations of metal ions, Examples of these assays are e.g. enzyme activity measurements and soil analysis with immunoassays where relatively high amounts of heavy metals may be present.
Mullinax et al. (U.S. Pat. No. 6,030,840, WO 99/66333) modified the DELFIA® method by adding some polyanionic compound to the enhancement solution. They teach that the dissociation of the lanthanide ion from the chelate is faster already at higher pH. The chelates used in their examples are a benzyl-EDTA derivative and DTPA where one acetate group is used for coupling to the biomolecule. The stabilities of these chelates are lower or about the same than those of the DTTA derivative used in the DELFIA® method. There is no proof or data that the method presented by Mullinax et al. would work with more stable chelates used for labeling of biomolecules. Compared to the commercialised enhancement solution (Wallac product), the above mentioned method does not provide improvement, also because the commercial DELFIA® enhancement already contains polyanions (phthalic acid).
Dakubu (U.S. Pat. No. 5,124,268) divided the fluorescence enhancement in two parts. The first part is the dissociation of the metal from the stable lanthanide chelate by lowering the pH to 1.5–3.0. The second part of the process is the change of the pH to over 3.5 and the development of the fluorescent lanthanide chelate. This two-step method is, however, too laborious to be used in automatic diagnostic systems because it demands one extra incubation and addition step.